Using genetics approaches to understand the structural basis for actin filament generation by the Arp2/3 complex ; The Arp2/3 complex is an actin nucleation factor conserved across evolution. In vitro, the Arp2/3 complex is activated by Wiskott-Aldrich syndrome family proteins and initiates the polymerization of a new filament at a distinct angle from an existing filament, resulting in a unique actin branch structure. These structures have also been observed in vivo and are thought to be critical for protrusive force generation during cell motility. The overall goal of this program project is to understand the detailed structural and biochemical mechanisms by which the Arp2/3 complex carries out its function. This project contributes genetic approaches to structure/function analysis of the Arp2/3 complex. Most of the aims involve using yeast genetic techniques to generate mutations or tags in various subunit of the Arp2/3 complex. Biochemical analysis and structural characterization (by NMR and electron cryo-microscopy, etc.) of the mutant or tagged complexes will allow testing specific models for Arp2/3 complex activation and actin nucleation, as well as defining the structural changes that occur during activation. In the last aim, we will use mouse embryonic stem cell genetics to generate cell lines bearing mutations in an Arp2/3 complex subunit that could affect actin branch formation, morphology or stability. Characterization of the dynamics and structure of branch junctions in fibroblasts differentiated from these cell lines wilt provide key insights into the role of the Arp2/3 complex in the generation and organization of actin filaments during cell motility.